Oxidative-Stress-Mediated ER Stress Is Involved in Regulating Manoalide-Induced Antiproliferation in Oral Cancer Cells

Manoalide provides preferential antiproliferation of oral cancer but is non-cytotoxic to normal cells by modulating reactive oxygen species (ROS) and apoptosis. Although ROS interplays with endoplasmic reticulum (ER) stress and apoptosis, the influence of ER stress on manoalide-triggered apoptosis has not been reported. The role of ER stress in manoalide-induced preferential antiproliferation and apoptosis was assessed in this study. Manoalide induces a higher ER expansion and aggresome accumulation of oral cancer than normal cells. Generally, manoalide differentially influences higher mRNA and protein expressions of ER-stress-associated genes (PERK, IRE1α, ATF6, and BIP) in oral cancer cells than in normal cells. Subsequently, the contribution of ER stress on manoalide-treated oral cancer cells was further examined. ER stress inducer, thapsigargin, enhances the manoalide-induced antiproliferation, caspase 3/7 activation, and autophagy of oral cancer cells rather than normal cells. Moreover, N-acetylcysteine, an ROS inhibitor, reverses the responses of ER stress, aggresome formation, and the antiproliferation of oral cancer cells. Consequently, the preferential ER stress of manoalide-treated oral cancer cells is crucial for its antiproliferative effect.


Introduction
Oral cancer is a severe type of cancer that is of increasing global concern [1]. The main factors correlated with mouth cancer include dangerous exposure to substance use, such as higher drinking, smoking, and betel nut chewing behaviors [2]. Additionally, viruses, fungus infections, nutritional deficiencies, radiation exposure, poor oral hygiene, and other chronic physical and chemical stimulations may be co-factors. In addition, gender, age, ethnicity, geographical culture, and lifestyle habits also affect the incidence of oral cancer [3]. There is also increasing research investment in the correlation between genes and environmental factors and the occurrence and deterioration of oral cancer [4]. Clinically, chemoradiotherapy and radiotherapy are used for oral cancer therapy, aside from surgery,

ER Expansion Change by Manoalide: Oral Cancer vs. Normal Cells
The changes in ER expansion patterns at different concentrations and exposure times of manoalide-treated oral cancer cells were assessed by flow cytometry (Figure 1A,C). Manoalide increased the intensity of ER (+) (%), namely ER expansion, in oral cancer cells   (Figure 1B,D). In contrast, ER expansion did not appear in normal cells (SG) because their ER staining intensities were similar to that of the control after manoalide treatment. Consequently, manoalide preferentially triggers ER expansion in oral cancer cells compared to normal cells.
Moreover, the influence of oxidative stress triggered by manoalide in controlling expansion was evaluated by the pretreatment of N-acetylcysteine (NAC) (Figure 1 Manoalide-induced ER expansion of oral cancer cells was downregulated by NAC ( Figu  1D). Hence, the role of ROS in manoalide-triggered ER expansion in oral cancer cells w confirmed.

Aggresome Change by Manoalide: Oral Cancer vs. Normal Cells
When ER stress is triggered, misfolded or denatured proteins are accumulated form aggresomes [31]. The changes in aggresome generation patterns at different conce trations and exposure times of manoalide treatment in oral cancer cells were assessed flow cytometry (Figure 2A,C). Manoalide increased the intensity of aggresome (+) (%) oral cancer cells ( Figure 2B,D). Consequently, manoalide preferentially triggers gresome formation in oral cancer cells compared to normal cells.
Moreover, the presence of NAC estimated the oxidative stress involvement in mo ulating aggresome generation ( Figure 2C). The manoalide-triggered aggresome accum lation of oral cancer cells was downregulated by NAC ( Figure 2D). Hence, the role of R in manoalide-triggered aggresome formation in oral cancer cells was confirmed. Moreover, the influence of oxidative stress triggered by manoalide in controlling ER expansion was evaluated by the pretreatment of N-acetylcysteine (NAC) ( Figure 1C). Manoalideinduced ER expansion of oral cancer cells was downregulated by NAC ( Figure 1D). Hence, the role of ROS in manoalide-triggered ER expansion in oral cancer cells was confirmed.

Aggresome Change by Manoalide: Oral Cancer vs. Normal Cells
When ER stress is triggered, misfolded or denatured proteins are accumulated to form aggresomes [31]. The changes in aggresome generation patterns at different concentrations and exposure times of manoalide treatment in oral cancer cells were assessed by flow cytometry (Figure 2A,C). Manoalide increased the intensity of aggresome (+) (%) in oral cancer cells ( Figure 2B,D). Consequently, manoalide preferentially triggers aggresome formation in oral cancer cells compared to normal cells.
Moreover, the presence of NAC estimated the oxidative stress involvement in modulating aggresome generation ( Figure 2C). The manoalide-triggered aggresome accumulation of oral cancer cells was downregulated by NAC ( Figure 2D). Hence, the role of ROS in manoalide-triggered aggresome formation in oral cancer cells was confirmed.

Modulation of ER Stress Signaling by Manoalide: Oral Cancer vs. Normal Cells
The mRNA expressions for ER stress signaling [32], including BIP, PERK, IRE1α, a ATF6, were assessed for manoalide treatment for 24 h in oral cancer cells and normal ce The relative mRNA expression of these ER-stress-associated genes (BIP, PERK, IRE1α, a ATF6) in oral cancer cells was generally higher than in the control, while it showed change in normal cells ( Figure 3A). BIP, IRE1α, and ATF6 genes were upregulated in o cancer (CAL 27) cells at a high dose (10 μM). IRE1α and ATF6 genes were overexpress in oral cancer (Ca9-22) cells at 5 and 10 μM of manoalide. In contrast, the mRNA expr sions of these ER-stress-associated genes of normal cells (SG) did not change.
Similarly, protein expressions for ER stress signaling were upregulated by manoali in different oral cancer cells ( Figure 3B). In CAL 27 cells, BIP, PERK, and IRE1α were mo erately upregulated, and ATF6 was slightly regulated. In Ca9-22 cells, only BIP and IRE were moderately upregulated. In comparison, the protein expressions of these ER str genes of normal cells (SG) were similar to the control and were lower than oral can cells. Notably, the moderately upregulated ER stress genes of manoalide treatment in o cancer cells were downregulated by NAC. Consequently, manoalide preferentially tr gers ER stress signaling in oral cancer cells compared to normal cells. Moreover, the r of ROS in manoalide-triggered ER stress signaling in oral cancer cells was confirmed.

Modulation of ER Stress Signaling by Manoalide: Oral Cancer vs. Normal Cells
The mRNA expressions for ER stress signaling [32], including BIP, PERK, IRE1α, and ATF6, were assessed for manoalide treatment for 24 h in oral cancer cells and normal cells. The relative mRNA expression of these ER-stress-associated genes (BIP, PERK, IRE1α, and ATF6) in oral cancer cells was generally higher than in the control, while it showed no change in normal cells ( Figure 3A). BIP, IRE1α, and ATF6 genes were upregulated in oral cancer (CAL 27) cells at a high dose (10 µM). IRE1α and ATF6 genes were overexpressed in oral cancer (Ca9-22) cells at 5 and 10 µM of manoalide. In contrast, the mRNA expressions of these ER-stress-associated genes of normal cells (SG) did not change.
Similarly, protein expressions for ER stress signaling were upregulated by manoalide in different oral cancer cells ( Figure 3B). In CAL 27 cells, BIP, PERK, and IRE1α were moderately upregulated, and ATF6 was slightly regulated. In Ca9-22 cells, only BIP and IRE1α were moderately upregulated. In comparison, the protein expressions of these ER stress genes of normal cells (SG) were similar to the control and were lower than oral cancer cells. Notably, the moderately upregulated ER stress genes of manoalide treatment in oral cancer cells were downregulated by NAC. Consequently, manoalide preferentially triggers ER stress signaling in oral cancer cells compared to normal cells. Moreover, the role of ROS in manoalide-triggered ER stress signaling in oral cancer cells was confirmed.

Antiproliferation by Manoalide and ER Stress Inducer: Oral Cancer vs. Normal Cells
To investigate the proliferation impact of manoalide-triggered ER stress, cells were co-treated with the ER stress inducer thapsigargin (TG), followed by manoalide treatment. Their modulating abilities on the proliferation of oral cancer cells and normal cells were examined by ATP analyses because ATP content is a common cell viability indicator [33,34]. Under ATP-content-based cell viability, manoalide showed a preferential antiproliferative effect on oral cancer cells, while it showed minor changes in normal cells ( Figure  4A). TG alone dramatically decreased the cell proliferation of oral cancer cells and slightly reduced it in normal cells. When the combined treatment of TG/manoalide was performed, the antiproliferative effect was enhanced in oral cancer cells. Furthermore, the combined treatment of manoalide/TG remains similarly viable to TG alone in normal cells (SG).
To explore the role of ROS, NAC was pretreated prior to the post-treatments of manoalide and/or TG in oral cancer cells and normal cells. Both ATP ( Figure 4B) and MTS ( Figure 4C) assays demonstrated the upregulated viabilities of manoalide and/or TG treatments in oral cancer cells upon NAC.

Antiproliferation by Manoalide and ER Stress Inducer: Oral Cancer vs. Normal Cells
To investigate the proliferation impact of manoalide-triggered ER stress, cells were co-treated with the ER stress inducer thapsigargin (TG), followed by manoalide treatment. Their modulating abilities on the proliferation of oral cancer cells and normal cells were examined by ATP analyses because ATP content is a common cell viability indicator [33,34]. Under ATP-content-based cell viability, manoalide showed a preferential antiproliferative effect on oral cancer cells, while it showed minor changes in normal cells ( Figure 4A). TG alone dramatically decreased the cell proliferation of oral cancer cells and slightly reduced it in normal cells. When the combined treatment of TG/manoalide was performed, the antiproliferative effect was enhanced in oral cancer cells. Furthermore, the combined treatment of manoalide/TG remains similarly viable to TG alone in normal cells (SG).
To explore the role of ROS, NAC was pretreated prior to the post-treatments of manoalide and/or TG in oral cancer cells and normal cells. Both ATP ( Figure 4B) and MTS ( Figure 4C) assays demonstrated the upregulated viabilities of manoalide and/or TG treatments in oral cancer cells upon NAC.

Modulation of Apoptosis by Manoalide and ER Stress Inducer: Oral Cancer vs. Normal Cells
The impact of ER stress on manoalide-induced apoptosis was assessed by caspase 3/7 analyses. TG activated more caspase 3/7 activity in oral cancer cells than in the control. Moreover, TG/manoalide activated more caspase 3/7 activity than manoalide alone in oral cancer cells (CAL 27 and Ca9-22), particularly at a high dose (10 μM) ( Figure 5). In contrast, the caspase 3/7 activity of manoalide treatment remained unchanged in normal cells (SG) with or without TG.

Modulation of Apoptosis by Manoalide and ER Stress Inducer: Oral Cancer vs. Normal Cells
The impact of ER stress on manoalide-induced apoptosis was assessed by caspase 3/7 analyses. TG activated more caspase 3/7 activity in oral cancer cells than in the control. Moreover, TG/manoalide activated more caspase 3/7 activity than manoalide alone in oral cancer cells (CAL 27 and Ca9-22), particularly at a high dose (10 µM) ( Figure 5). In contrast, the caspase 3/7 activity of manoalide treatment remained unchanged in normal cells (SG) with or without TG.

Modulation of Apoptosis by Manoalide and ER Stress Inducer: Oral Cancer vs. Normal Cells
The impact of ER stress on manoalide-induced apoptosis was assessed by caspase 3/7 analyses. TG activated more caspase 3/7 activity in oral cancer cells than in the control. Moreover, TG/manoalide activated more caspase 3/7 activity than manoalide alone in oral cancer cells (CAL 27 and Ca9-22), particularly at a high dose (10 μM) ( Figure 5). In contrast, the caspase 3/7 activity of manoalide treatment remained unchanged in normal cells (SG) with or without TG.

Modulation of Autophagy by Manoalide and ER Stress Inducer: Oral Cancer vs. Normal Cells
The impact of ER stress on manoalide-induced autophagy was assessed by DAPGreen (DAP) [35] and DALGreen (DAL) [36] analyses. Manoalide induced more DAP (+) and DAL (+) levels in oral cancer cells than in the control ( Figure 6A,B). Moreover, TG/manoalide induced more DAP (+) and DAL (+) levels than manoalide alone in oral cancer cells (CAL 27 and Ca9-22) ( Figure 6A,B). In contrast, the DAP (+) and DAL (+) levels of manoalide treatment remained unchanged in normal cells (SG) with or without TG. ± SDs (n = 3). Multiple comparisons were analyzed for the same cell lines. Treatments marked with different notes indicate a significant difference (p < 0.05 to 0.0001).

Modulation of Autophagy by Manoalide and ER Stress Inducer: Oral Cancer vs. Normal Cells
The impact of ER stress on manoalide-induced autophagy was assessed by DAPGreen (DAP) [35] and DALGreen (DAL) [36] analyses. Manoalide induced more DAP (+) and DAL (+) levels in oral cancer cells than in the control ( Figure 6A,B). Moreover, TG/manoalide induced more DAP (+) and DAL (+) levels than manoalide alone in oral cancer cells (CAL 27 and Ca9-22) (Figure 6A,B). In contrast, the DAP (+) and DAL (+) levels of manoalide treatment remained unchanged in normal cells (SG) with or without TG.

Discussion
We previously found that manoalide provided preferential killing and the induction of oxidative stress, DNA damage, and apoptosis of oral cancer cells, while it showed minor changes to normal cells [29,30]. However, the interplay of ROS and apoptosis with ER stress [37] and the function of ER stress in the manoalide-caused antiproliferative effect of oral cancer were not investigated. The present study assessed the ER stress responses/signaling and apoptosis by modulating ROS or ER stress in the manoalide treatment of oral cancer cells and normal cells.

Discussion
We previously found that manoalide provided preferential killing and the induction of oxidative stress, DNA damage, and apoptosis of oral cancer cells, while it showed minor changes to normal cells [29,30]. However, the interplay of ROS and apoptosis with ER stress [37] and the function of ER stress in the manoalide-caused antiproliferative effect of oral cancer were not investigated. The present study assessed the ER stress responses/signaling and apoptosis by modulating ROS or ER stress in the manoalide treatment of oral cancer cells and normal cells.
As detected by flow cytometry, manoalide at a high dose (10 µM) induced higher ER expansion and aggresome accumulation of oral cancer cells than normal cells (Figures 1  and 2). These ER stress phenotypes were further examined by analyzing the mRNA and protein expression of ER stress signaling genes (Figure 3). CAL 27 and Ca9-22 cancer cells showed a higher mRNA expression of BIP, IRE1α, and ATF6 genes than the untreated control, particularly at 10 µM manoalide, but these were slightly changed in normal cells. In comparison, manoalide did not induce PERK mRNA expression in oral cancer cells.
Similarly, the protein expressions of BIP, PERK, IRE1α, and ATF6 genes of oral cancer cells and normal cells showed a similar tendency in their mRNA expressions. Notably, these ER-stress-associated proteins provided lower levels in normal cells than in oral cancer cells. This suggests that manoalide shows preferential induction of ER stress signaling in oral cancer, while it shows little changes in normal cells. However, the current study did not examine the impact of ER stress signaling on manoalide-triggered responses. It warrants a thoughtful assessment of knockdown BIP and/or IRE1α to see if the knockdown alters the outcomes of studies concerning viability, ER expansion, and apoptosis in the future.
Prolonged exposure to manoalide causes higher ER expansion and aggresome accumulation of oral cancer cells than normal cells, reversed by NAC. Similarly, some ER-stressassociated genes showing higher mRNA and protein expressions in oral cancer cells following manoalide treatment were reversed by NAC. This suggests that manoalide-triggered ER stress and its signaling are ROS-dependent. The role of ROS acting on apoptosis has been reported in our previous work [29,30], indicating that manoalide triggers apoptosis of oral cancer cells depending on ROS levels. Moreover, ER stress [21] and ROS [38] may trigger autophagy. This warrants a thorough investigation of the relationship between ROS, apoptosis, autophagy, and ER stress.
ER stress inhibits proliferation and triggers apoptosis of cancer cells [39][40][41][42]. TG, an ER stress inducer, suppresses the proliferation of breast cancer cells, and the combined treatment of TG with the engineered fusion protein (epidermal growth factor-proteolytic A subunit) enhances its antiproliferative effect [39]. TG also causes apoptosis of adrenocortical carcinoma [40] and prostate [41] cancer cells. Similarly, TG induces higher antiproliferation and apoptosis (caspase 3/7 activation) of manoalide-treated oral cancer cells than the control (Figures 4 and 5). Like TG, manoalide triggers ER stress and apoptosis of oral cancer cells.
Furthermore, the impact of ER stress on manoalide-triggered antiproliferation, apoptosis, and autophagy was assessed in the present study. TG further enhances manoalideinduced antiproliferation, apoptosis (caspase 3/7 activation) (Figures 4 and 5), and autophagy ( Figure 6). In the example of hepatocytes, TG induces ER stress and antiproliferation, reversed by NAC [43], suggesting that TG triggers ROS induction to promote ER stress and antiproliferation. NAC can reverse the manoalide-induced antiproliferation [29] and ER stress responses (Figures 1-3) of oral cancer cells. Similarly, NAC effects on the viability of TG/manoalide treatment of oral cancer cells were validated in the present study, demonstrating that TG/manoalide synergistically inhibited proliferation in an ROS-dependent manner ( Figure 4B,C). Moreover, TG/manoalide synergistically triggered apoptosis ( Figure 5), which may enhance antiproliferation. TG/manoalide also synergistically triggered autophagy ( Figure 6); however, the role of autophagy in the antiproliferation of manoalide-treated oral cancer cells remains unclear. Consequently, manoalide exhibits the interplay of ER stress with ROS, apoptosis, and the preferential antiproliferation of oral cancer, but they are slightly changed in normal cells.
Besides apoptosis and autophagy, drug-induced ER stress may modulate and interact with several cellular functions, such as ferroptosis. For example, C2-ceramide triggers autophagy of liver cancer cells by upregulating oxidative stress and ER stress [44]. Notably, several anti-tumor drugs may exhibit the interplay between apoptosis and autophagy under ER stress [45][46][47]. Autophagy inhibitors promote apoptosis of prostate cancer cells triggered by overexpressing melanoma differentiation-associated gene 7 (mda-7) [48]. In contrast, downregulated apoptosis enhances autophagy after mda-7 overexpression. The relationship between ER-stress-modulated apoptosis and autophagy in manoalide-treated oral cancer cells may be explored by choosing their modulators. Moreover, ferroptosis triggers ER stress by interacting with apoptosis [49]. It warrants a thoughtful investigation for manoalide-induced other ER-stress-associated cell functions of oral cancer cells in the future.

Cell Culture and Chemicals
We chose the oral cancer cell lines (CAL 27 and Ca9-22) from the JCRB and ATCC for this study. As for normal oral cell line, we chose Smulow-Glickman (SG), a human normal gingival epithelial cell line that is well characterized [50,51] and commonly used to test drug safety for dental materials [52,53] and anti-oral-cancer drug development [54,55]. CAL 27/Ca9-22 and SG cells were cultured using mixtures of Dulbecco's Modified Eagle Medium (DMEM) and F12 (Gibco, Grand Island, NY, USA) at ratios of 3:2 and 4:1 [29], respectively, with mixtures of 10% serum and common culture antibiotics.

ER Expansion
ER content was stable, but it increased (namely ER expansion) in response to ER stress. Using the Organelle-ID RGB ® III Assay Kit (Enzo Life Sciences, Farmingdale, NY, USA) [60], the degree of ER expansion was monitored by flow cytometry. Briefly, cells were mixed with ER staining dye under incubation for 30 min at 4 • C. After washing with the medium twice, cells were stood for 30 min at 37 • C and analyzed using the Guava easyCyte flow cytometer (Luminex, Austin, TX, USA), plotting with FlowJo software (Becton-Dickinson, Franklin Lakes, NJ, USA).

Aggresome
Upon ER stress, aggresomes commonly increase. Aggresomes were stained using Proteostat ® Aggresome Detection dye (Enzo Life Sciences) [61], and the degree of aggresome accumulation was determined by flow cytometry. Briefly, cells were prefixed with 4% paraformaldehyde for 30 min and permeabilized by 0.5% Triton X-100 for 30 min at 4 • C. Finally, cells were stained with aggresome staining dye (1:10,000) for 30 min, analyzed using a Guava easyCyte flow cytometer and plotted with FlowJo.

mRNA Expressions of ER-Stress-Associated Genes
RNA was extracted for cDNA conversion [62]. Quantitative RT-PCR (qRT-PCR) running with a touch-down PCR program was performed as described [62]. The mRNA expressions of four ER-stress-associated genes were analyzed [32], such as BIP, PERK, IRE1α, and ATF6. Their primer information is provided in Table 1. Glyceraldehyde 3phosphate dehydrogenase (GAPDH) was the control gene [63,64]. The fold activation of mRNA expressions was evaluated by 2 -∆∆Ct calculations [65].

Statistics
One-way ANOVA with Tukey's HSD post-hoc test was performed by JMP ® 12 (SAS Institute Inc., Cary, NC, USA) to determine the significances between multiple comparisons. Treatments of non-overlapping notes showed significant results.

Conclusions
Manoalide, characterized originally as an antibiotic, was here repurposed for anticancer treatments and the preferential antiproliferation and apoptosis of oral cancer cells was monitored, while it showed little impact on normal cells [29,30]. However, ER stress commonly interplayed with apoptosis, but the involvement of ER stress with oral cancer cells following manoalide treatment has rarely been investigated.
The present study validates that manoalide triggers more ER stress in oral cancer cells than in normal cells. ER expansion and the aggresome accumulation and gene expressions of ER-stress-associated proteins were upregulated by manoalide in oral cancer cells, but they were slightly changed in normal cells. Moreover, the potential connections between ER stress, antiproliferation, apoptosis, and autophagy were explored. Utilizing ER stress inducer TG, the manoalide-triggered antiproliferation and apoptosis were further enhanced. This finding indicates that manoalide-triggered ER stress is vital in controlling antiproliferation, apoptosis, and autophagy of oral cancer cells, but they showed low changes in normal cells. Consequently, manoalide showed preferential ER stress induction and contributes to the preferential antiproliferation of oral cancer cells.